Recently there has been considerable focus in the conjugation of oligonucleotides with non-nucleotide moieties which add additional functionality to the oligonucleotide, for example, enabling targeting of therapeutic oligonucleotides to specific organs and tissues in vivo. For targeting to liver hepatocytes trivalent GalNAc conjugation moieties capable of binding to asialoglycoprotein receptors have been found to allow for vastly reduced dosages whilst delivering effective therapeutic action, see for example WO2014/118267. WO 2013/033230 also describes a variety of oligonucleotide conjugates including antibodies, carbohydrates, cholesterol, insulin, PEG, transferrin and vitamins. The manufacture of oligonucleotide conjugates is therefore of major importance, particularly when considering the increased costs of manufacture of the conjugate group and the additional process steps in conjugation.
Milesi et al., Methods in Enzymology (1999) 313, pp 164-173, reports on the synthesis of oligonucleotide conjugates in anhydrous dimethyl sulfoxide. The procedure used by Milesi et al. uses triethylammonium salt to solublise an 10 bp DNA oligonucleotide in DMSO, prior to conjugation to a lipophilic 5′minor groove binding conjugate, and is recommended for use with lipophilic and hydrostatically unstable groups. According to Milesi et al., 2.5 molar equivalents of conjugate group were used as compared to the oligonucleotide, and typically up to 20 molar equivalents of the conjugate group are used.